Throughout this application various publications are referred to in superscripts. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The generation of recombinant deoxyribonucleic acid (DNA) molecules is an essential tool in modern molecular biology. The conventional DNA cloning strategies that have been used for several decades typically involve the use of type II restriction enzymes to generate appropriate DNA fragments, the modification of DNA ends to generate blunt or sticky ends and the ligation of the DNA fragments to generate plasmid or other type DNA vectors1-3. However, these procedures depend on the presence of appropriate restriction sites to generate both vector and insert molecules and often leave unwanted sequences at the junction sites. In addition, the restriction enzymes and modifying enzymes required for these manipulations are often expensive making these procedures costly especially in high throughput settings. The present invention addresses the need for a seemless and restriction site-independent cloning method.